SELF-REGULATING BIOADHESIVES FOR WET SUBSTRATES
Herein disclosed is a mucoadhesive including a bacterial cellulose, and a bioadhesive incorporated to the bacterial cellulose, wherein the bioadhesive comprises a polymer or a dendrimer, wherein the polymer and the dend rimer are grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus. A method of forming the mucoadhesive is also disclosed. The method includes providing a bacterial cellulose, providing a bioadhesive comprising a polymer or a dendrimer, wherein the polymer and the dendrimer are grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus, and incorporating the bioadhesive to the bacterial cellulose. In the preferred embodiment, the dendrimer grafted with the moiety is Poly(amidoamine) (PAMAM) dendrimer grafted with 4-[3-(trifluoromethyl)-3H-diazirin-3-yl] benzyl bromide (PAMAM-g-diazirine) and the stimulus is UV light.
This application claims the benefit of priority of Singapore Patent Application No. 10202011922U, filed 30 Nov. 2020, the content of it being hereby incorporated by reference in its entirety for all purposes.
TECHNICAL FIELDThe present disclosure relates to a mucoadhesive. The present disclosure also relates to a method of forming the mucoadhesive.
BACKGROUNDThe Global Burden of Disease Study 2017 estimated that about 45% of the world population is affected by non-communicable oral diseases, which include a series of chronic diseases affecting the oral cavity and may manifest in the form of wounds and ulcers. The oral cavity presents challenges like high bacterial and microorganisms load, presence of saliva and salivary enzymes, wet environment due to constant salivary flow, soft mucosal tissues, and dynamic shear forces that prevent implant fixation and subsequent treatment. Therefore, materials and adhesives have been generated specifically for soft tissue fixation within the buccal and gastrointestinal regions, which may primarily consist of mucous membranes on epithelial surfaces. The materials and adhesive may be referred to as mucoadhesives, and may consist of topical formulations of semi-solid, liquid, or film-based formulations.
Existing therapies based on plasters or mucoadhesives are limited in effectiveness due to adhesive failure (in less than 24 hrs), loss of structural integrity, or a combination thereof. Alternatives may include frequent applications (3-5/day) of liquid or gel, leading to poor patient compliance. Commercial mucoadhesive designs are based on water swelling polymers that stick to soft tissues via polymer entanglement at the tissue surface. The design is relatively safe, but the uncontrolled water absorption leads to worsening adhesion strength over time (0.5-5 kPa, assuming area of 2 cm2). Hence, the mucoadhesive's structures have a limited fixation of 6-8 hours. Mucoadhesive designs may be improved if swelling plateaued, thus preventing cohesive and adhesive failure.
According to one example, current mechanisms of buccal mucoadhesion may rely on hydrophilic polymers that absorb water on the buccal tissues. Hydrophilic attractive forces result from mucoadhesive polymers entangling macromolecules on the tissue surface (e.g. glycoproteins, mucin, extracellular matrix proteins). The mucoadhesive forces may withstand the physiological shear forces for only a limited length of time. The primary reason for adhesion failure may be due to uncontrolled absorption of water, which decreases the number of entanglements, weakens the cohesive forces within the mucoadhesive, decreases the hydrophilic attractive forces, or a combination thereof. After a critical amount of water absorption, adhesion forces may be overcome by physiological shear forces and delamination, disintegration, fracture, or a combination thereof. Currently available adhesives may not adequately address uncontrolled water absorption, do not provide covalent crosslinking to tissue surfaces, or allow predetermined periods of fixation.
Current mucoadhesives based on water swelling, hydrophilic polymers are activated when applied to wetted tissue surfaces. They are reported to adhere non-specifically to wet surfaces with weak adhesion stresses ranging from 1-5 kPa (assuming an area of 2 cm 2). The main limitation is the limited adhesion time of 6-8 hours. Overhydration tends to be responsible for the short retention periods. Treatments based on mucoadhesives with extended fixation times of 2-7 days may target unmet clinical needs, however, mucoadhesives which typically rely on reversible attractive forces such as hydrogen bonds, electrostatic interactions, and van der Waals bonds, may not allow fixation of medical implants for 2-7 days.
There is thus a need to provide for a solution that addresses one or more of the limitations mentioned above. The solution should at least provide for a mucoadhesive that resolves one or more of the limitations mentioned above and a method of forming such a mucoadhesive.
SUMMARYIn a first aspect, there is provided a mucoadhesive including:
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- a bacterial cellulose; and
- a bioadhesive incorporated to the bacterial cellulose,
- wherein the bioadhesive comprises a polymer or a dendrimer, wherein the polymer and the dendrimer are grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus.
In another aspect, there is provided a method of forming the mucoadhesive described in various embodiments of the first aspect, the method includes:
-
- providing a bacterial cellulose;
- providing a bioadhesive comprising a polymer or a dendrimer, wherein the polymer and the dendrimer are grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus; and
- incorporating the bioadhesive to the bacterial cellulose.
The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the present disclosure. In the following description, various embodiments of the present disclosure are described with reference to the following drawings, in which:
The primary adhesion mechanism provided by PDz is due the ability to form carbenes upon UV exposure. In case of BC_PDz composites, it is hypothesized that these carbenes would react majorly with the —OH groups and minorly with —CH— groups present in the bacterial cellulose structure as depicted in
The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the present disclosure may be practised.
Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.
The present disclosure relates to a mucoadhesive and method of forming the mucoadhesive. The present mucoadhesive and method relates to an approach through covalent bonding that may provide enhanced adhesion strength and retention on a tissue surface. Film release then becomes dependent on natural epithelial sloughing, where complete renewal of the oral epithelium surface may occur over 5 days. This is advantageous over current mucoadhesives, which traditionally may be hydrogels that rely on weak forces for soft tissue fixation, for example, hydrogen bonds and van der Waals forces in order of decreasing bond strength.
The present mucoadhesive and method involves bacterial cellulose and a bioadhesive. As the present mucoadhesive is not composed of a single component but formed from multiple components, the present mucoadhesive may be herein interchangeably referred to as a “bioadhesive composite”, “bioadhesive platform”, “composite” and “system”.
Bacterial cellulose (BC) is a naturally derived hygroscopic polymer which may be obtained from Acetobacteraceae family members, which absorbs 80-100 times water of its own dry weight. However, unlike other water swelling macromolecules, hydration reaches a plateau with retention of material properties. This inherent self-regulating water absorption ability of BC can be advantageous for moist environments like the oral cavity. The nanoporous structure provides a barrier with translucent properties for light transmission. Other industrial-friendly attributes include its production in the metric tons and edible nature attesting to low biotoxicity risks. It is sold commercially as a food product, nata de coco, justifying its safe use. BC-based wound dressings are also approved by FDA specifically for burns owing to its hydration and barrier properties.
The present mucoadhesive and method involve a bioadhesive. In various embodiments and examples herein, poly(amido amine) (PAMAM) dendrimer grafted with aryl-diazirine was utilized as one non-limiting example to demonstrate for the present bioadhesive platform for investigating covalent bonding of surface proteins upon wet surfaces. Diazirine generates reactive carbene crosslinking group upon activation with UVA (365 nm) or voltage. Various PAMAM-g-diazirine (PDz) formulations incorporating natural and synthetic polymers have resulted in tunable shear moduli of 1-250 kPa and maximum shear adhesion strength of 5-200 kPa when attached to collagen matrix or ex vivo tissue surfaces. However, PDz does not control for water swelling, but when PDz is integrated with BC (BC_PDz), the resultant bioadhesive platform has improved properties to handle water swelling. Hence, the present mucoadhesive may also be termed herein a “BC_PDz composite” or “BC_PDz adhesive”. Apart from PDz, other bioadhesives that can form covalent bonding with a wet tissue surface can be used, e.g., those containing a moiety which generate a carbene upon activation.
The present mucoadhesive is assessed for enhanced wet soft mucosal tissue adhesion. The combination of self-regulating water absorption of BC, a bioadhesive that can form covalent bonds with a wet tissue surface (e.g. carbene-based crosslinking), and cohesive strength retention, yields a composite for adhesion to a variety of buccal and gastrointestinal ailments. Performance assessment prioritizes the structure activity relationship between ultraviolet A (UVA) energy and hydration state on lap shear adhesion strength. In the present disclosure, the hydration state may be controlled via two processes, i.e., freeze-drying and press-drying. Both processes advantageously retain the properties of BC without shrinkage, which allows for manageable film thicknesses between 30-250 μm, suitable for oral cavity applications, to be produced.
Details of various embodiments of the present mucoadhesive and advantages associated with the various embodiments are now described below. Where the advantages are already demonstrated in the examples further hereinbelow, they shall not be iterated for brevity.
In the present disclosure, there is provided a mucoadhesive. The mucoadhesive includes a bacterial cellulose and a bioadhesive incorporated to the bacterial cellulose. The bioadhesive can include a polymer or a dendrimer. The polymer and the dendrimer may be grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus. The tissue surface may be a wet tissue surface, e.g. containing water or moisture. The tissue surface herein refers to any surface of a biological tissue.
In various embodiments, the polymer may include polycaprolactone, polyethylenimine, polyester, chitosan, etc. Other polymers which do not hinder covalent interactions with the tissue surface may be suitable.
In various embodiments, the dendrimer may include a poly(amidoamine) or polyethylenimine. A “dendrimer” is distinct from other classes of polymers (including the polymer mentioned hereinabove) in that the dendrimer has a plurality of dendritic chains emanating radially from a central core unit as compared to a polymer merely having entities extending from a polymer backbone wherein the moieties are not radially emanating therefrom. The term “poly(amidoamine)” may be herein abbreviated as “PAMAM”. The poly(amidoamine) may be a G5-PAMAM dendrimer (5th generation, molecular weight (Mw)=28.8 kDa)
In various embodiments, the moiety may include a diazirine. In various embodiments, the moiety may be a moiety capable of forming covalent interactions with a tissue surface via carbene-based crosslinking. In certain non-limiting embodiments, the moiety may include a diazirine, and the polymer or the dendrimer may include polycaprolactone or a poly(amidoamine) dendrimer, respectively.
In certain non-limiting embodiments, the moiety may include an electron donating moiety capable of oxidizing into a ketone (e.g. a monoketone or diketone) when subject to a stimulus. Such electron donating moiety may include, and is not limited to, 1,2-aryl-diol (e.g. a catechol or a catechol variant (e.g. a triol)), 1,4-aryl-diol, a vanillin, a derivative thereof, etc. In this instance, the stimulus may include water (e.g. moisture, water containing oxygen, saliva, mucus an oxidant, an electrical voltage, or a combination thereof. In instances where the mucoadhesive contains such a moiety and/or is activated by water, such a mucoadhesive may be termed herein “water-activated mucoadhesive”. In certain non-limiting embodiments, the electron donating moiety may be attached directly on an electron accepting moiety that is in turn directly on the polymer and/or the dendrimer. The electron accepting moiety advantageously gets reduced as the electron donating moiety gets oxidized for cross-linking and renders the present adhesive composition operable as an adhesive. The electron accepting moiety may include, and is not limited to, an imine, an alkene, an oxadiazole, a tetrazinealkyne, a fluoren-9-one, a thioazole, or a malononitrile.
In various embodiments, the bacterial cellulose may be a film including a network of cellulose nanofibers. In various embodiments, the bacterial cellulose may be dried or rehydrated.
In various embodiments, the bioadhesive may be deposited on the bacterial cellulose. In certain non-limiting embodiments, the bioadhesive may be deposited solely on the bacterial cellulose. Said differently, having the bioadhesive deposited on the bacterial cellulose means that the bioadhesive is present only on the surface of the bacterial cellulose, i.e. not present within the bacterial cellulose (the bacterial cellulose may be internally absent of the bioadhesive).
In certain non-limiting embodiments, the bacterial cellulose may be a hydrogel. In embodiments where the bacterial cellulose is a hydrogel, the bioadhesive may be present in an amount of 10 wt % to 35 wt %, 10 wt % to 33.3 wt %, 20 wt % to 33.3 wt %, etc. of the mucoadhesive.
In certain non-limiting embodiments, the bioadhesive may be included in the bacterial cellulose. In certain non-limiting embodiments, the bioadhesive may be included in the bacterial cellulose and may be present on the surface of the bacterial cellulose. In other words, the bioadhesive may be present within the bacterial cellulose and externally on the bacterial cellulose. In certain non-limiting embodiments, the bioadhesive may be solely within the bacterial cellulose, i.e. absent on the surface of the bacterical cellulose or externally absent on the bacterial cellulose. In certain non-limiting embodiments, the mucoadhesive may be an integrated patch wherein the bioadhesive is included within the bacterial cellulose. In embodiments where the bioadhesive is present within the bacterial cellulose, e.g. the integrated patch, the bioadhesive may be present in an amount of 15 wt % to 60 wt %, 20 wt % to 40 wt %, etc. of the mucoadhesive.
In various embodiments, the bacterial cellulose is of a species derived from a family of Acetobacteraceae (Acetobacteraceae family), which include and is not limited to, Acetobacter, Gluconacetobacter, and/or Komagataeibacter species.
In various embodiments, the bacterial cellulose may be optically pervious to light having a wavelength ranging from 280 nm to 1100 nm.
In various embodiments, the stimulus may be moisture, a chemical (e.g. water with or without oxygen), a biological fluid (e.g. saliva, mucus), an electrical and/or an optical stimulus. In various embodiments, the stimulus may include light having a wavelength ranging from 280 nm to 500 nm, and/or a voltage ranging from −10 kV to 10 kV. Advantageously, the present optical stimulus of using light having such wavelengths avoid cancer-causing wavelengths of 100 nm to 250 nm.
The present mucoadhesive may further include a pharmaceutical drug incorporated to the mucoadhesive. The pharmaceutical drug may include peptide or protein drugs. In certain non-limiting embodiments, the pharmaceutical drug may be incorporated to the bacterial cellulose. Non-limiting examples of the pharmaceutical drug include lidocaine, dexamethasone and valganciclovir. The present mucoadhesive may further include a polymeric coating, for example, on the bacterial cellulose. Non-limiting examples of the polymeric coating include polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA). The polymeric coating may reside on any surface of the bacterial cellulose. Said differently, the polymeric coating may be configured between the bacterial cellulose and the bioadhesive, such that the bioadhesive is deposited on the polymeric coating. In certain instances, the polymeric coating may reside on the bioadhesive, for example, the polymeric coating may reside on one side of the bioadhesive and the bacterial cellulose resides on the opposing side of the bioadhesive away from the polymeric coating. This configuration may be denoted as, for example, BC//bioadhesive//polymeric coating.
The present disclosure also relates to a method of forming the mucoadhesive described in various embodiments of the first aspect. The method may include providing a bacterial cellulose, providing a bioadhesive including a polymer or a dendrimer, wherein the polymer and the dendrimer are grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus, and incorporating the bioadhesive to the bacterial cellulose. Embodiments and advantages described for the present mucoadhesive of the first aspect can be analogously valid for the present method subsequently described herein, and vice versa. As the various embodiments and advantages have already been described above and demonstrated in the examples further hereinbelow, they shall not be iterated for brevity.
In various embodiments, providing the bacterial cellulose may include inoculating bacteria in one or more culture media, extracting raw bacterial cellulose from the one or more culture media, and/or treating the raw bacterial cellulose with an alkali to remove the bacteria from the bacterial cellulose.
In various embodiments, providing the bacterial cellulose may include freeze-drying or press-drying the bacterial cellulose.
The present method may further include rehydrating the bacterial cellulose which is freeze-dried or press-dried prior to incorporating the bioadhesive.
In various embodiments, providing the bioadhesive may include grafting a diazirine on a polycaprolactone or a poly(amidoamine) dendrimer, and mixing the polycaprolactone or the poly(amidoamine) dendrimer grafted with the diazirine in a buffer. Other suitable polymers or dendrimers and moieties as described in various embodiments of the mucoadhesive of the first aspect may be used.
In certain non-limiting embodiments, incorporating the bioadhesive to the bacterial cellulose may include depositing the bioadhesive on the bacterial cellulose. In certain non-limiting embodiments, the bioadhesive may be deposited solely external of the bacterial cellulose. Said differently, the bioadhesive may be absent within the bacterial cellulose and deposited only on the surface of the bacterial cellulose.
In certain non-limiting embodiments, incorporating the bioadhesive to the bacterial cellulose may include contacting the bacterial cellulose with an organic solvent containing the bioadhesive, and removing the organic solvent to have the bioadhesive included in the bacterial cellulose. This may help in forming the integrated patch mentioned above in certain non-limiting embodiments of the mucoadhesive of the first aspect.
In certain non-limiting embodiments, incorporating the bioadhesive to the bacterial cellulose may include mixing the bacterial cellulose and the bioadhesive to form a hydrogel.
In various embodiments, providing the bacterial cellulose may include mechanically or chemically treating the bacterial cellulose to render cellulose nanofibers. For example, providing the bacterial cellulose may include sonicating the bacterial cellulose, which helps in the forming of the hydrogel.
The present method may further include a polymeric coating on the bacterial cellulose.
In summary, the present BC-based mucoadhesive is biocompatible and edible which makes it a good candidate for oral applications. BC has FDA GRAS status. Hence, the present BC-based mucoadhesive may allow for localized and extended delivery of therapeutics to the target sites in the oral cavity, i.e. an approach that addresses (prevents) uncontrolled absorption of moisture, provides attractive forces, covalent bonding, or combinations thereof, which would lead to mucoadhesive fixation for predetermined periods. Advantageously, due to the better adhesion from covalent bonding and control over water absorption, prolonged retention can be achieved on a wet tissue surface which in turn confers increased contact time at the oral lesion, extending therapeutic doses and providing a wound barrier.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the present disclosure.
In the context of various embodiments, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements.
In the context of various embodiments, the punctuation “—”, term “about” and “approximately” as applied to a numeric value encompasses the exact value and a reasonable variance.
As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
Unless specified otherwise, the terms “comprising” and “comprise”, and grammatical variants thereof, are intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, unrecited elements.
EXAMPLESThe present disclosure relates to a self-regulating mucoadhesive and a method of forming thereof. The present mucoadhesive provides numerous advantages over existing mucoadhesive formulations.
Traditionally, topical approaches to oral diseases may require frequent dosing due to limited wound retention times. The present mucoadhesive serves as a drug delivery platform applicable to oral wound sites with soft tissue adhesion capability of up to 7 days. The present mucoadhesive and method involve, as non-limiting examples, bacterial cellulose (BC) and photoactivated carbene-based bioadhesives combined to yield a flexible film platform for interfacing soft tissues in dynamic, wet environments. Structure-activity relationships are evaluated via UV dose and hydration state with respect to adhesive strength on soft tissue mimics. The present mucoadhesive, which may be exchangeably termed herein “bioadhesive composite”, can have an adhesion strength ranging from 7-17 kPa and duration exceeding 48 hours in wet conditions under sustained shear forces. This is advantageous over mucoadhesives based on hydrophilic macromolecules which unfortunately exhibits adhesion strength of 0.5-5 kPa and last only a few hours. The examples hereinbelow describe evaluations of the mucoadhesive treatments in the buccal cavity.
The present mucoadhesive and method are described in further details, by way of non-limiting examples, as set forth below.
Example 1A: General Discussion on Method of Present Bioadhesive Production Including Bacterial Cellulose Production and CharacterizationExamples 1A to 1C are generally intended to demonstrate non-limiting examples of the composition and utility of bacterial cellulose (BC) and the present photoactivated bioadhesive for adhesion to wet and buccal tissue surfaces. BC is used herein as a self-regulating, water-absorbing polymer. PAMAM-g-Diazirine (PDz) bioadhesive provides covalent bonding to wet tissue substrates but has limited adhesion strength to buccal tissues due to fouling by the immobilized water layer. Water competes with macromolecule covalent bonding. BC removes and absorbs water from the tissue surface, increasing the probability of covalent interactions between bioadhesive and tissue grafted macromolecules (e.g., glycoproteins, proteins within the extracellular matrix).
BC films are produced using 5% inoculum of Gluconacetobacter hansenii 53582 sp. in 2 ml HS (Hestrin-Schramm) media inoculated in 24 well plates. The films produced after 5 days of culture were used for further studies. The produced wet films weighed around 400-600 mg and the diameter of the films was found to be around 1.5±0.1 cm. In this study, freeze-drying (BC_FD films, 160 μm thickness) and press-drying (BC_PD films, 30 μm thickness) methods are employed to obtain dry BC films as they do not require any chemicals, help retain the properties of BC without shrinkage of nanofibers, and produce films with thickness 30-250 μm suitable for oral cavity applications.
Grafting of diazirine to PAMAM backbone is performed. In brief, a PAMAM solution in methyl alcohol (0.5% w/v) was prepared and diazirine was added directly into 5 mL of the PAMAM solution (according to theoretical calculations). The conjugation reaction was set for 36 hours with stirring under room temperature. Methyl alcohol was removed by vacuum and PAMAM-g-diazirine was collected as a pale yellow viscous liquid. PAMAM-g-diazirine with 20% conjugation and 50% concentration in PBS is employed for further experiments. BC_PDz composites are formed by adding varying moles of PDz to BC, either in film or hydrogel form as depicted in
This example demonstrates the BC films exhibit UV dose based tunable adhesion with strength of 6-22 kPa against wet porcine tissue.
The adhesion strength of photocured BC films with PDz bioadhesive is determined using lap shear test against porcine cheek skin as illustrated in
It is found that BC films exhibit tunable adhesion based on the type of BC film (BC_PD, BC_FD), hydration state (dry, rehydrated) and UV dose. The average adhesion strength for BC_FD for varying applied UV dose ranges from 5.9-8.3 kPa for dry films and 7.3-14 kPa for rehydrated films (
Diazirine functionalized PLGA films have also been evaluated for adhesion to tissues in wet environment with average lap shear adhesion strength of 4.5 kPa. Injectable mucoadhesive system based on chitosan grafted dihydrocaffeic acid and oxidized pullulan have been reported to exhibit average adhesion strength of 5 kPa. Maximum adhesion strengths of previously reported film based mucoadhesive systems have been reported to be 0.65 kPa for polyacrylates based system, 0.2-0.55 kPa (assuming 2 cm2 film area) for chitosan and hydroxyethylcellulose blend, 1-2.7 kPa for Eudragit®, carboxymethylcellulose (CMC) and hydroxymethylcellulose blends, 4-10 kPa for polyvinylpyrrolidone (PVP) and CMC blend and 0.5-2 kPa (assuming 2 cm2 film area) for various natural gum based films. Herein, it is demonstrated higher adhesion strength ranging from 6-22 kPa exhibited by BC films adhered with the aid of PDz bioadhesive compared to existing film based adhesive systems.
Example 1C: Extended Duration of Fixation within Wet Environments Under Physiological StressesThe present example involves an ex vivo mucoadhesion study that reveals adhesion time of 2-7 days in viscous aqueous environment.
The present example aims to investigate the adhesive behaviour of BC films with PDz in aqueous environment for long durations.
Similar synthetic electrospun nanofiber membranes formed of dextran and poly(ethylene oxide) as adhesive agents have been reportedly evaluated for their potential as mucoadhesive membranes for targeting oral cavity. The membranes exhibited in vitro and ex vivo mucoadhesion time of 200-250 minutes (4 hours approximately). Another study reported synthesis of mucoadhesive hydrogels wherein catechol was covalently bonded to chitosan further crosslinked with genipin exhibiting in vitro mucoadhesion time of 6 hours. Another reported study described the synthesis of sulfhydryl anchored adhesive alginate for management of oral aphthous stomatitis which exhibited mucoadhesion time of 100 minutes. The frequency of currently marketed mucoadhesives as mentioned in
Examples 2A to 2T are generally intended to demonstrate non-limiting examples of the present mucoadhesive and method for oral cavity applications.
Polyamidoamine (PAMAM) dendrimer, Generation 5, Mw=28.8 kDa, was purchased from Dendritech, Inc., USA and 3-[4-(bromomethyl) phenyl]-3-(trifluoromethyl)-diazirine(bromodiazirine) from TCI, Japan. All other chemicals were purchased from Sigma-Aldrich unless specified otherwise.
Strains and culture medium: G. hansenii 53582 ATCC was utilized for the fermentation of BC in Hestrin Schramm (HS) medium consisting of glucose (20 g L−1, 0.11 M), citric acid monohydrate (1.15 g L−1, 0.005 M), Na2HPO4 (2.7 g L−1, 0.02 M) enriched with yeast extract (5 g L−1) and peptone (5 g L−1) with final pH 4.5. HS media is prepared and autoclaved at 120° C. for 15 min.
Example 2B: Microbial FermentationFor the production of inoculum, bacterial strain Gluconacetobacter hansenii ATCC 53582 is inoculated in the culture media (HS Media) for 24 hours in shaking condition at 26° C. G. hansenii is later inoculated in 5% HS basic medium and cultured in 24-well plates at 26° C. for 5 days. The obtained BC pellicles are treated with sodium hydroxide solution (0.25 N) for 30 minutes at 60° C., and then washed thoroughly with deionized water for removal of bacterial cells (confirmed with protein determination—see example 2C). The films are then dried using lyophilizer (Alpha 2-4 LSC basic freeze dryer) which are later denoted as Freeze-dried BC (BC_FD). The other set of films are dried under pressure using bloating paper and designated as Press-dried BC (BC_PD). Both kinds of films are evaluated for mechanical properties (Mechanical Tester Instron 5567—see example 2D), light transmission properties (see example 2E) and structural morphology (Scanning electron microscopy, JEOL JSM-6700 FESEM) (see example 2F).
Example 2C: Determination of Protein Content in BC Films300 mg of BC native pellicle corresponding to 3.9 mg of dry BC is extracted in 20 ml of PBS buffer by sonication. The PBS extract is assayed for protein content using Thermo Scientific™ Micro BCA™ Protein Assay Kit. The microplate reader procedure with working range of 2-40 μg/ml is used for the assay. The protein concentration in extract was found to be 9.3±0.2 μg/ml. Thus, the total protein extracted is (9.3×20) 186 μg, which is approximately 48 μg/mg of dry cellulose.
Example 2D: Tensile Test of BC FilmsThe tensile strength of the FD, FD Rh, PD and PD Rh BC films is determined using Instron 5567 Electromechanical Tester. The films of rectangular shape with average gauze length of 7 cm and width of 1.5 cm according to ASTM standards are prepared. The thickness of freeze-dried and press-dried films was determined before the experiment. Tension Test Method was used for determining the tensile strength with load cell of 500 N and Tensile Extension of 3 mm/min. Rate of Load (50% Sensitivity) was set as the end of test criteria. The results were expressed as average of 3 test specimens and shown in
The light transmission properties of different kinds of BC films is determined using the setup shown in
The structural morphology of dried BC_FD and BC_PD films is observed using field emission scanning electron microscope (JEOL, JSM-6700 F, Japan) operating at an accelerating voltage of 5 kV and 8 mm working distance. The samples are staged on stubs with the help of double-sided carbon tape, and sputter coated with platinum for 70 s, 20 mA current in vacuum (8-9 Pa) before observation.
Example 2G: Synthesis of PAMAM-g-diazirineGrafting of diazirine to PAMAM backbone is performed as described in example 1A. PAMAM-g-diazirine with 20% conjugation and 50% w/w concentration in phosphate buffered saline (PBS) with pH 7.2, is employed for further experiments.
Example 2H: SEM ImagingBC_PDz films adhered to PET sheets are cured at UV dose of 18 J cm−2 (365 nm, 100 mW·cm−2). After curing, the PET sheet is peeled away to expose the cohesively fractured surface of PDz. The structural morphology of these fractured surfaces is observed using field emission scanning electron microscope (JEOL, JSM-6700 FESEM) operating at an accelerating voltage of 5 kV and 8 mm working distance. The samples are staged on stubs with the help of double-sided carbon tape, and sputter coated (JEOL JFC-1600 Auto Fine Coater) with platinum for 70 s, 20 mA current in vacuum (8-9 Pa) before observation. Image J software is employed to determine the pore length and pore number.
Example 21: Lap Shear Adhesion Assessment Against Porcine TissuesPorcine jowl skin (2×1.5 cm2, 0.2 cm thickness) obtained from local suppliers and BC films (2 cm 2) are subsequently mounted onto glass slides using cyanoacrylate glue. PDz (˜4 mg, 30 μm thickness) sandwiched between BC films and porcine tissue is photocured (365 nm, 100 mW·cm−2) for 30 s, 60 s, or 180 s. Adhesion strength test of BC_PDz films is determined by a modified tensile tester (Chatillon force measurement, Largo, FL, USA) with constant linear speed of 3 mm/min with 50N loading cell (n=5).
Example 2J: FTIR Analysis of Cured and Uncured BC_PDz FilmsFTIR (PerkinElmer Frontier) measurements are performed with Universal ATR fixture of diamond crystal. Photocured and uncured BC films with PDz are placed directly onto the crystal. The samples are scanned over a range of 4000 to 600 cm−1 with a resolution of 4 cm−1 and presented as an accretion of 32 scans.
Ex vivo mucoadhesion time is assessed for BC films with aid of PAMAM-g-diazirine (PDz). The porcine tissue (2×2 cm2) is attached to the internal side of the beaker using cyanoacrylate glue, and the BC films are adhered to tissue with the aid of PDz. The beaker is filled with 50 ml of the 50 mM phosphate buffer (pH 6.8) with magnetic stirring rate of 200-300 rpm accounting for constant shear stress of approximately 0.072-1.08 Pa per minute. The duration of film adhesion is observed during a period of 2 days under solutions with apparent viscosities of 1, 10, and 30 mPa·s. The viscous liquids are composed of 0, 2 and 3% w/v with 20-40 kDa sodium alginate respectively. An extended analysis is performed with rabbit tongue (fresh tongue tissue of rabbit carcass donated from other PI project from SingHealth Experimental Medicine Centre, Singapore) and 1 mPa·s solution for 7 days period.
Measurement of shear stress and normal force on the tissue substrate were performed using a beaker of radius 2 cm as shown in
Shear rate=velocity/height;
Shear stress=viscosity*shear rate;
ω is the angular velocity calculated from the recorded rpm. The bead rpm is 24 for 1 cps fluid, 36 for 10 cps and 6 for 30 cps fluid.
Placing in the values here, the shear stress at the wall was found to be 0.072 Pa for 1 cps, 1.08 Pa for 10 cps and 0.54 Pa for 30 cps approximately every minute.
The friction coefficient of tongue against the soft tissues in the oral cavity is found to be 0.1-0.3.
Friction coefficient=Shear force/Normal force
Thus, placing the values of friction coefficient and shear force in the above equation, the normal force on the BC_PDz film adhered to the tissue at the wall of beaker would be around 0.01-0.2 N, equivalent to gram force of 1-22. (wherein in stress=Force/Area).
Example 2L: Statistical AnalysisAll the data are presented in the form of mean±SD (n=3, unless otherwise stated). OriginPro 2018 64-bit software is employed to determine significance using Two-way ANOVA with Bonferroni as post-hoc test. A p<0.05 (*) is considered statistically significant.
Example 2M: Discussion of Results for Oral Cavity ApplicationsBC pellicles are obtained after 5 days of inoculation of G. hansenii under static conditions, and further washed (0.25 N NaOH, deionized water) to obtain native BC pellicle (
The pore size and density are determined by scanning electron microscopy (SEM) after photocuring. Surface chemistry is qualitatively evaluated with infrared spectroscopy to correlate functional groups with material properties. A final ex vivo mucoadhesion test in submerged saline is performed to evaluate the holding strength under constant shear stress for up to 7 days.
Example 2N: Discussion of Results for Oral Cavity Applications—BC Transparency Increases Upon HydrationAs photocuring is dependent on the UV light transmission through the BC films, the optical properties require moderate light transmission. The mass of a 16-mm diameter native BC pellicle is 860±40 mg (
For the determination of swelling ratio of BC films, the dried films (BC_FD, BC_PD) are weighed and noted as H D. The weighed films are then immersed in 5 ml of phosphate buffer saline (pH 7.4) at room temperature for 3 hours. The swollen films are then re-weighed and noted as Hs. The swelling ability of the films is calculated using the formula:
Swelling Ratio=(HS−HD)/HD (S2)
Swelling Percentage=(HS−HD)*100/HD (S3)
Electron micrographs of BC_FD and BC_PD surfaces reveal that freeze-dried films retain their nanofiber networks and nanoporous structure, which collapsed in press-dried films (
Photocuring adhesives are typically activated by UVA with wavelengths ranging from 350 to 405 nm and thus require sufficiently transparent film for its activation. The experiment is conducted using representative UV LEDs of 365 nm, 385 nm, and 405 nm that spanned the range of photocuring wavelengths required of both diazirine and free-radical based bioadhesives. An increase in light transmission is observed with hydration, but no clear correlation is observed with hydration percentage. Not surprisingly, transparency also increases with respect to longer wavelengths due to reduced scattering. The BC_FD films exhibit maximum light transmittance at 200% hydration at 365 nm (required for PDz activation), while that for BC_PD films is at 100% hydration (not significantly different from 200%, p>0.05). Thus, 200% hydration is chosen for the remaining studies. The detailed method for measurement of light transmittance through BC films is already described in example 2E above.
Example 20: Discussion of Results for Oral Cavity Applications—BC/PDz Composite Adhesives Exhibit 7-17 kPa Lap Shear Adhesion Strength Against Ex Vivo Porcine TissueConstant salivary flow is expected to lead to instant hydration of films upon application to the diseased oral sites. The hydration of films affects the optical properties and subsequently might affect the adhesion properties. To examine the effect of hydration on adhesion performance, the films are evaluated in dry and rehydrated states. Hydration of the films led to variations in the light transmission properties of films (see
The adhesion strength of photocured BC films is determined using lap shear test against porcine cheek skin as illustrated in
The top left and top right plots in
Mean adhesion strength for BC_PD films for varying UV doses ranges from kPa which is almost double the adhesion strength exhibited by BC_FD (5.9-8.3 kPa) films in dry state, however not significantly different. The bottom right plot of
Surface morphology of the uncured and cured films is observed using SEM. Variations in terms of pore length and density are analyzed.
FTIR analysis of BC films with PDz before and after curing aids in the qualitative analysis of changes in the functional groups. Reactions with the —OH and —CH— groups present in the BC structure are monitored before and after UVA exposure. The FTIR spectra for BC, PDz, uncured BC_PDz film, and photocured BC_PDz film are displayed in
In order to challenge the BC_PDz films, the photocured adhesives are exposed to constant shear aqueous environments for 2 days on porcine tissues. To further stress the films, hypotonic 50 mM phosphate buffer pH 6.8 is employed. The dried BC_PD films are applied with PDz on the porcine tissue fixed on the beaker wall and activated using UVA (365 nm, 100 mW·cm−2, 180 s) directly from the BC film side placed on the tissue. Later, the beaker is filled with fluids of varying viscosities, 1, 10, and 30 cps, to simulate the range of human saliva viscosity. The process is similar to clinicians' procedure of applying medication to oral tissue, which is later hydrated due to salivary release. BC_PD films are chosen in this experiment owing to their low swelling ability, thus, to study the matrix stability of thin films upon prolonged aqueous exposure. The beaker in
Overcoming weak adhesion strength (0.5-5 kPa) and short retention durations (6-8 hours) in wet oral environment is a current unmet clinical need in the treatment of oral wounds. Frequent applications lead to reduced patient compliance. Thus, systems that offer adhesion strength sufficient for prolonged retention durations and maintain structural integrity in the wet environment may improve therapeutic outcomes. Towards addressing this limitation, bacterial cellulose (BC) films were paired with carbene-based bioadhesives with the aim of long-term retention on hydrated soft mucosal tissue surfaces. To the best of our knowledge, this is the first demonstration of BC with covalent bond formation as the mechanism of adhesion. The system consists of two independent components to yield a synergistic composite: (1) BC film (freeze-dried, FD, and press-dried, PD) and (2) PDz (G5-PAMAM grafted with diazirine). BC films undergoing different drying processes exhibit different mechanical, morphological, and light transmission properties (
BC serves as the structural component within the composite, providing barrier and mechanical properties of the film. In contrast to plant cellulose, BC biosynthesized by aerobic bacterium Gluconacetobacter family members is pure and yields a form that prevents overhydration. Cellulose obtained from plant sources contains impurities, requires processing to produce nanofibrillated cellulose, has low surface area, and poor light transmission. Hydrophilic cellulose derivatives utilized as mucoadhesives undergo swelling in contact with water leading to dissolution/disintegration of the matrix. BC extends the opportunity to utilize pure cellulose in the form of nanofibrous thin films that exhibit selfregulated swelling in aqueous environment while not being dissolved/eroded off. The intrinsic nanofiber matrix provides a high surface area for interactions with drug/active molecules and light transmission properties. Two methods of film post-processing are demonstrated. Freeze-drying helps to maintain the 3D structure of BC film but is likely to be an energy-intensive manufacturing practice. On the other hand, press-drying allows in-line manufacturing through industry standard calendaring operations with the tradeoff of compacted 3D network and dense aggregation of nanofibers as visible in
Hydrated BC films exhibit lower tensile strength, increased elongation, and higher modulus of elasticity (0.04-1 GPa, similar to reported traditional non-BC films) compared to the dry state as observed in
Drying process also affects rehydration ability (59% for BC_FD, 4% for BC_PD) and transparency of the films (
PDz bioadhesives are activated using light/voltage to bond wet tissue surfaces (adhesion strength 6-27 kPa) by means of covalent bonds. Surface conforming PDz gel composites have been evaluated for adhesion strength with various macromolecules, including hydrophilic PEG400 (5-15 kPa), hydrophobic polycaprolactone alcohols (5-kPa), and alginate (8-10 kPa). PDz has also been evaluated on film substrates like PLGA (4.5 kPa) and polymer brush coated glass (15-40 kPa). Herein, the present example demonstrated similar adhesion strength ranging from 7-17 kPa exhibited by BC_PDz film systems that combine the advantages of gels (conforming, water absorption) and films (strength). The thin, flexible BC matrix allows conformal contact with the tissue surface, thereby overcoming limitations offered by stiffer, hydrophobic substrates like PLGA. Moreover, the BC matrix does not disintegrate in aqueous environments (
The presence of PDz on BC surface leads to pore formation at the interface upon photocuring, as observed in
Hydrophilic water swelling macromolecules based on natural, synthetic polymers and their blends have been widely employed in mucoadhesive systems. Maximum adhesion strengths of mucoadhesive films have been reported to be 0.65 kPa for polyacrylates, 0.2-0.55 kPa (assuming 2 cm2 film area) for chitosan and hydroxyethylcellulose (HEC) blend, 1-2.7 kPa for Eudragit®, carboxymethylcellulose (CMC) and hydroxymethylcellulose (HMC) blends, 3 kPa (assuming 2 cm 2 film area) for electrospun chitosan membranes with pectin coating and 0.5-2 kPa (assuming 2 cm2 film area) for various natural gum (alginate, xanthan gum, CMC, HEC) based films evaluated using normal/tensile test. Injectable mucoadhesive based on chitosan grafted dihydrocaffeic acid and oxidized pullulan exhibited average adhesion strength of 5 kPa and polyvinylpyrrolidone (PVP), CMC blend films exhibited 20 kPa as evaluated by lap shear test. Electrospun nanofibrous meshes composed of poly(l-lactic acid) and mucoadhesive poly(ethylene oxide) exhibited adhesion strength ranging from 1.5-3 kPa (assuming 2 cm 2 film area). The lap shear tests exhibit about 2-5 times higher adhesive strength than normal/tensile test. Taking that into consideration, the average adhesion strength for these mucoadhesives relying on hydrogen bonds ranges from 0.5-5 kPa.
Desired properties of mucoadhesives in aqueous oral cavity environment include mucoadhesion time (>24 hours) and matrix stability. These properties are required to maintain extended contact with the diseased site. However, current systems exhibit mucoadhesion residence less than 24 hours. Carbopol 934, ethylcellulose blend (12 hours), blend of HPMC, Eudragits, Carbopol 940 (1.5-2 hours), chitosan/gelatin blend (6 hours), sulfhydryl anchored adhesive alginate (1.5 hours) and xanthan gum blends (1-5 hours) exhibit average mucoadhesion time of 1.5-6 hours. Other mucoadhesives like electrospun nanofiber membranes (polycaprolactone, dextran, polyethylene oxide) and catechol-chitosan based mucoadhesive hydrogels exhibited in vitro and ex vivo mucoadhesion time of 4-6 hours. BC_PDz patch exhibits ex vivo mucoadhesion time of 2-7 days under shear stress, but this does not take into account the dynamic environments within the oral cavity, e.g., tissue strains, ionic effects, friction. This relatively low adhesion strength (0.5-5 kPa) and adhesion time (<24 h) of existing mucoadhesives are limited in effectiveness due to weak adhesion forces (hydrogen bonds, van der Waals bonds), loss of structural integrity, or combination thereof. The present BC_PDz system addresses these limitations in two ways; first, the swelling of the composites plateau due to self-regulating water absorption property of BC, thus maintaining structural integrity in wet environments (
Several advantages of the present mucoadhesive are reiterated. The BC_PDz mucoadhesive system provides light tunable adhesion with higher adhesion strength (7-17 kPa) and extended ex vivo mucoadhesion time (2-7 days) compared to existing systems. Distinct from other types of reported films, hydration of BC films retain the adhesivity, i.e., the films exhibit comparable adhesion strength in both wet and dry states. The BC_PDz system exhibits cohesive failure upon removal thereby the assurance of no tissue damage upon adhesive patch removal. However, the system exhibits certain limitations. In the present example, PDz was applied each time to BC film before application, wherein clinical application need a shelf-stable dry composite. Cohesive failure within the PDz matrix highlights the stresses are not distributed across the BC matrix, suggesting poor integration of the globular PDz and nanofibrous BC.
Composites of bacterial cellulose and carbene-based bioadhesives are herein demonstrated to adhere directly to tissue substrates in the oral cavity. Bacterial cellulose films serve as the structural component capable of loading active pharmaceutical ingredients, and a surface layer of carbene-based bioadhesives provides light activated, on-demand adhesion. Tunable adhesion strength ranging from 7-17 kPa against porcine tissue with cohesive failure is obtained (compared to 0.5-5 kPa of cellulose derivative-based systems). The composite adhesion strength correlates to the UV dose applied and the hydration state. The composite patch withstood 7 days in a submerged aqueous environment under constant shear stress when applied to ex vivo tissue substrates. The present composite demonstrates a new platform towards mucoadhesive delivery systems for the management of oral cavity diseases and lesions.
Example 2T: Drug Release Profile of Adhesive Patches (BC_PDz Composite)The present example demonstrates that adhesive patches loaded with protein drug exhibits tunable drug release behaviours (24 hrs). The adhesive patches in this instance are BC_PDz composite. The adhesive patches were loaded with bovine serum albumin (BSA), a model protein drug (66.5 kDa) with the drug loading efficiency as mentioned in
Topical approaches to oral wounds and infections exhibit weak adhesion to wet surfaces, short retention duration (6-8 hours), frequent dosing requirement and patient incompatibility. To address these, film based adhesive platforms comprising of bacterial Cellulose (BC) and photoactive carbene-based bioadhesive (PDz) exhibiting adhesion strength of 7-17 kPa against wet tissues was demonstrated in examples 2A to 2S. However, films may be less suitable (albeit still applicable) for application to deep irregular shaped wounds, gap filling, and diseases widespread in the oral cavity. Thus to further extend the applicability of BC_PDz systems, photoactive aqueous adhesive hydrogels are formulated. Structure-activity relationships evaluate BC mole, bioadhesive moles, and total solute percentage on viscoelastic properties and wet tissue adhesion. The aqueous composites crosslink upon photocuring in 15-70 seconds and exhibit transition from viscous to elastic state gradually. The photoactive composites have shear moduli ranging from 1-10 kPa, thereby mimicking oral mucosa. The composites exhibit on-demand tunable adhesion strength of 7-22 kPa in wet environments, similar to previously reported BC film systems. The work highlights the potential of BC/PDz hydrogel systems for treatment of diseases in the wet environment of oral cavity.
In more details, examples 2A to 2S demonstrated the combination of Bacterial cellulose (BC) films with PAMAM-g-diazirine (PDz) adhesives exhibiting tunable adhesion strength of 7-17 kPa against wet tissues. The idea was to utilize stronger covalent bonds formed by carbenes with mucosal proteins to adhere BC films to oral mucosa in place of weak hydrogen and van der Waals bonds. The epithelial sloughing process responsible for epithelium renewal every 5-6 days can lead to release of the bonded system. PDz bioadhesive chemically constitutes of Poly(amido amine) (PAMAM) dendrimer grafted with 4-[3-(trifluoromethyl)-3H-diazirin-3-yl] benzyl bromide(diazirine) and has been evaluated for adhesion to wet tissue surfaces. Diazirine groups present in the PDz generate reactive crosslinking groups (carbenes) upon photocuring or electorcuring as demonstrated earlier. The photocured PDz composites have been demonstrated to exhibit tunable shear moduli of 1-250 kPa and maximum adhesion strength of 5-45 kPa against wet tissue substrates. However, the film-based BC systems are less suitable (albeit still applicable) for applications in cases such as widespread disease sites over oral cavity area, irregular shaped wounds/lesions, gap filling, etc. Thus, to further widen the applicability of BC based adhesive systems, an aqueous composite hydrogel of BC and PDz which can be applied/smeared to the irregular shaped diseased site was developed. The applied adhesive would turn into a viscoelastic solid upon photocuring thereby offering tunable shape property. BC is a naturally derived hygroscopic polymer from Gluconacetobacter species, which exhibits controlled hydration with retention of material properties. This inherent self-regulating water absorption ability of BC can be advantageous for moist environments like oral cavity. High storage modulus of nanofibrous BC hydrogel is proposed to be utilized to improve the material properties of BC_PDz composites upon photoactivation. Carbenes formed upon photocuring PDz has been demonstrated to react easily to —OH groups and form solid matrix. Similar reaction is expected in case of exposure of carbenes to —OH abundant BC hydrogel resulting in formation of viscoelastic solid. Moreover, the incorporation of BC may further lead to reduced concentration of cationic amine groups thereby increasing cytocompatibility. BC is sold commercially as a food product, nata de coco, attesting to low biotoxicity risks and ease of large scale industrial production of BC. Wound dressings based on BC for burns and other topical wounds have also been approved by FDA owing to its hydration properties.
Based on the abovementioned properties of PDz and BC, it has been demonstrated that composites of BC and PDz may exhibit higher shear moduli than BC or PDz alone. It is also demonstrated that these composites can further exhibit synergistic wet tissue adhesion (lap shear adhesion). Structure-activity relationships of BC_PDz aqueous composite is evaluated for dynamic viscosity, photocuring rheology, morphological analysis, crosslinking mechanism, and lap shear adhesion strength. The effect of total solute percentage, BC moles, and PDz moles in the composites upon mechanical properties and adhesion strength exhibited by BC_PDz composites is studied.
Example 3B: Materials and Method for Example 3AMaterials: Polyamidoamine (PAMAM) dendrimer, Generation 5, Mw=28.8 kDa is bought from Dendritech, Inc, USA. 3-[4-(bromomethyl) phenyl]-3-(trifluoromethyl)-diazirine (bromo-diazirine) is purchased from TCI, Japan. All other chemicals were purchased from Sigma Aldrich, unless specified otherwise.
Strains and culture medium: G. hansenii 53582 ATCC is employed for the fermentation of BC in Hestrin Schramm (HS) media. HS is media composed of glucose (20 g L−1, 0.11 M), citric acid monohydrate (1.15 g L−1, 0.005 M) and Na2HPO4 (2.7 g L−1, 0.02 M) enriched with yeast extract (5 g L−1) and peptone (5 g L−1) with final pH 4.5. Media is prepared and autoclaved at 120° C. for 15 mins.
Microbial fermentation: Bacterial strain Gluconacetobacter hansenii ATCC 53582 is inoculated in the culture media (HS Media) for 24 hours in shaking conditions at 26° C. to produce the inoculum. 5% (v/v) of the G. hansenii inoculum is inoculated in HS basic medium and cultured in 24-well plates at 26° C. for 5 days. The BC pellicles are harvested and washed with distilled water followed by sodium hydroxide treatment (0.25 N) for 30 minutes at 60° C. for removal of bacterial cells (confirmed with protein determination as described in example 2C). The pellicles are then washed thoroughly with deionized water. BC produced and harvested after 5 days is further employed for formulating defibrillated dispersion.
Synthesis of PAMAM-g-diazirine: Grafting of diazirine to PAMAM backbone is performed as described in the above examples. PAMAM-g-diazirine with 20% conjugation is employed for further experiments.
Formulation of BC_PDz hydrogels: BC_PDz composites are formulated as depicted in
Real-time photorheometry evaluation of BC_PDz composites: Rheometry measurements are conducted with Anton Paar Physica MCR 102 rheometer fitted with parallel plate probe PP10 and UVA transparent glass slide, as presented in
SEM imaging: BC_PDz composites are placed between PET films and photocured with UVA dose of 10 J cm−2 (365 nm, 100 mWcm−2). After photocuring, the PET sheets are peeled apart to expose the cohesively fractured composite matrix to analyze the structural morphology. A field emission scanning electron microscope (JEOL, JSM-6700 F, Japan) (accelerating voltage of 5 kV and 8 mm working distance) is employed to observe the structural morphology of the cured BC_PDz composites. The samples are sputter coated (JEOL JFC-1600 Auto Fine Coater) with platinum for 70 s, 20 mA current in vacuum (8-9 Pa) before imaging. Image J software is employed to determine the pore length and pore number per unit area. The scale bar is utilized as a measurement reference, and the longest length on the pore is recorded.
Lap shear adhesion assessment against PLGA and wet tissue mimicks: PLGA films (2×1.5 cm 2) and wet tissue mimic (hydrated collagen films, 2×1.5 cm 2) are fixed on the glass slides using double sided adhesive tape. BC_PDz composites (10 mg, 100 μm thickness) are then smeared in between the films and activated by UVA (365 nm, 100 mW cm−2) for 100 seconds (10 J UV dose). The adhesion strength of the formulated BC_PDz composites is determined using modified tensile tester (Chatillon force measurement, Largo, FL, USA) with a controlled linear speed of 3 mm/min with 50N load cell (n=5). PLGA films are cast using 30% (w/v) PLGA (50:50) solution in DCM (stirred overnight to aid dissolution). The viscous solution is then cast onto flat glass substrate using a knife applicator at 50 mm/sec at room temperature in fume hood. The cast PLGA films are dried at room temperature overnight in the fume hood to prevent foaming. Films are then transferred to vaccum oven and kept for one week for solvent evaporation at 37° C. Thickness of PLGA film was found to be 0.06±0.0039 mm.
Statistical analysis: All the data are presented as mean±SD (n=3, unless otherwise stated). OriginPro 2018 64-bit software is employed to determine significance employing Two-way ANOVA with Bonferroni as posthoc test. A p<0.05 (*) is considered statistically significant. Rheology and lap shear adhesion curves are smoothed to remove transient noise by using percentile filter and Loess (span 0.1) method, respectively.
Example 3C: Discussion of Results for Example 3A—Formulation of BC_PDz Hydrogels, Investigation of Structure-Activity Relationship with PhotorheologyAqueous adhesive composites are produced by mixing varying moles of Bacterial Cellulose (BC) and PAMAM-g-diazirine (PDz) as per the schematic in
The total solute percentage of aqueous adhesives is varied from 10% to 33.3%. The percentage of PDz which is the major component is varied from 9-32.5%. In the BC film based platform of examples 2A to 2S, the concentration of PDz bioadhesive was 50%. However, reduced tissue exposure of cationic —NH2 groups in PDz may help avoid mammalian cell cytotoxicity. To address this issue, lower concentrations of PDz (9-32.5%) are evaluated in the present compositions. Real-time photorheology is performed to examine the degree of crosslinking of BC_PDz composites under UVA exposure. The corresponding changes in the storage modulus (G′) and loss (G″) moduli are recorded in real-time using a customized photorheometry setup described in
Thereafter, the variation in the photocuring behavior of composites with the variation of BC moles for 20 and 33.3% total solute is determined. BC moles are varied from 0-80 millimoles.
In case of 33.3% total solute photorheograms presented in
Thus, the BC_PDz hydrogels exhibit storage modulus of 1-10 kPa with crosslinking time varying from 15-70 seconds and light dose of 1.5-7 J. The crosslinking time, light dose and light intensity required for photocuring of the composites fall within the general curing protocols employed in dentistry, which varies from 5-100 seconds with light intensity ranging from 300-2000 mW cm−2, dose of 8-50 J/cm2 for lights with 350-500 nm wavelengths.
Example 3E: Discussion of Results for Example 3A—Increase in BC Content and Total Solute Percentage have Opposite Effects on Yield Stress and Strain Values for the BreakThe third phase of photorheology consists of amplitude sweeps ranging from 1-1000% strain performed after photocuring as depicted in
The setup of lap shear adhesion test of photocured BC_PDz composite performed against PLGA and wet tissue mimic hydrated collagen is illustrated in FIG. top left image. The composites placed in the sandwich structure are exposed to UVA (365 nm, 100 mW cm−2, 10 J UV dose).
The representative stress-strain profile obtained by the lap shear test of BC_PDz composites placed between PLGA films is presented in
Overall, the composites exhibit adhesion strength ranging from 10-55 kPa against PLGA substrates and 7-35 kPa against wet tissue mimic. The adhesion strength is lower in the case of wet tissue mimics which is expected due to reaction with water molecules.
From the examples above, it can be seen that the present aqueous BC_PDz adhesive hydrogels display higher storage modulus and adhesion strength compared to non-aqeuous PDz composites with higher PDz concentration.
PAMAM-g-diazirine was demonstrated as a liquid adhesive hydrogel for application in minimally invasive surgery. The shear moduli of the formulations and the adhesion strength were found to be varying with the percentage of diazirine conjugated to the PAMAM dendrimer, the concentration of the PDz in aqueous media and UV dose. For the PDz formulation with 20% conjugation, the lowest concentration evaluated was 25% which upon photocuring was found to exhibit storage modulus ranging from 1-3 kPa (measuring gap 0.1 mm, 6 J UV dose). The adhesion strength for the same formulation against wet tissue was found to be around 5 kPa. Formulations with 20% diazirine conjugation and higher concentrations (50 and 75%) exhibited higher shear moduli ranging from 20-200 kPa and adhesion strength ranging from 25-35 kPa. However, there are several limitations associated with the presence of high concentrations of PDz. One of the limitations is higher foaming caused due to the nitrogen release that occurs due to higher PDz concentration in the formulation, thereby causing a trade-off between adhesion strength and porous nature. Another concern may be the biocompatibility of the bioadhesive systems, due to the presence of cationic —NH2 groups. Several attempts have been made to overcome these limitations by replacement of the aqueous solvent of PDz bioadhesive by hydrophilic PEG400 in one case and hydrophobic polycaprolactone alcohols in the other. The lowest concentration of PDz present in non-aqueous PEG/PDz composites was found to be 30% with an approximate storage modulus of 25 kPa after photocuring for applied UV doses of 10, 20 and 60 J cm−2 (0.1 mm measuring gap). The examples also suggested that a negative correlation exists between effective storage modulus after photocuring and the initial measuring gap. The adhesion strength of 30% PDz composites against tissue was found to be ranging from 5-10 kPa. Similarly, the PDz/PCLT composites with concentration of PDz as low as 20 and 33.3% were formulated and found to exhibit G′ maximum of approximately 0.1 kPa and 4 kPa respectively (measuring gap 0.1 mm, 20 J UV dose). The adhesion strength of these composites against wet tissue mimic was found to be ranging from 5-15 kPa for 20% concentration and 5-25 kPa for 33.3% concentration. For the dry substrate, adhesion strength for 20% concentration ranged from 1-15 kPa, and for 33.3%, it ranged from 1-10 kPa. Another composite of PDz where alginate was incorporated exhibited storage modulus of 30 kPa (measuring gap 0.1 mm, 20 J UV dose) and adhesion strength of 8-10 kPa against wet substrates.
In examples 3A to 3G, the formulation and evaluation of the aqueous composites of BC and PDz for its mechanical properties and adhesion to wet as well as dry substrates are demonstrated. Composites with low PDz concentrations, i.e. 20 and 33.3% with varying BC moles have been evaluated. The composite comprises (1) BC hydrogel, which improves the mechanical properties (2) and PDz, which is PAMAM grafted with diazirine responsible for adhesion to wet surfaces by carbene insertion mechanism. The composites exhibited an increase in the dynamic viscosity depending on the PDz as well as BC moles. Moreover, the composites exhibit shear moduli ranging from 1-10 kPa in positive correlation with BC moles and percentage of total solute (measuring gap 0.45 mm). The composites thereby mimic the shear moduli of oral mucosal tissues. Taking into consideration discussed studies about negative correlation of measuring gap with shear moduli, the BC_PDz aqueous composites exhibit comparable shear moduli to non-aqueous PDz/PCLT and PDz/PEG composites even when measured at four times higher measuring gap. Moreover, the aqueous BC_PDz composites with about 20% and 33.3% PDz exhibit adhesion strength of 8-40 kPa and 22-70 kPa respectively, which is comparatively 2-3 times higher than that exhibited by PDz/PCLT composites with dry substrates. Similarly, composites with about 20% and 33.3% PDz exhibited adhesion strength of 3-33 kPa and 8-30 kPa respectively with hydrated tissue mimic, which is again 2-3 times higher than PDz/PEG, PDz/PCLT and PDz/alginate composites.
Inclusion of nanofibrous BC into the composite allows for crosslinking at lower PDz concentrations and exhibit higher storage modulus mimicking oral cavity tissues.
Bacterial Cellulose is one of the varieties of cellulose, most commonly synthesized by aerobic bacteria Gluconacetobacter species. BC includes a nanofibrous matrix with complex molecular structure wherein —OH groups of cellulose form hydrogen bond with inter and intra-molecular glucan chains. The nanofibrous matrix of BC dispersion is presented in
BC has been found to exhibit linear viscoelastic region only up to 6-7% of strain as observed from
SEM micrographs exhibit trade-off between pore formation and adhesion strength upon photocuring dependent on PDz concentration.
SEM images presented in
BC_PDz adhesive composites display lower crosslinking duration and higher adhesion strength onto wet tissue surface mimic compared to other mucoadhesives.
The BC_PDz composites evaluated herein are demonstrated for adhesion to the oral cavity for applications like localized drug delivery, adhering drug loaded films to disease site and as protective coating onto diseased/wound surfaces. Currently employed mucoadhesives exploit hydrogen bond interactions formed by polymers as the adhesion mechanism, resulting in low adhesion strength (8-50 Pa). Mucoadhesive films based on chitosan, hydroxypropylmethylcellulose (HPMC), and xanthan gum loaded with lidocaine hydrochloride have been evaluated for periodontal systems and found to exhibit low adhesion strength of 8-16 Pa (assuming area of 3 cm2). A reported emulsion-based mucoadhesive system for oral candidiasis was found to exhibit average adhesion strength of 33 Pa (assuming area of 3 cm2). Another report evaluated a different mucoadhesive system composed of Eudragits (a mixture of methyl and ethyl acrylates), and HPMC loaded with Fluconazole with 50 Pa maximum adhesion strength (assuming area of 3 cm2) for application in oral candidiasis. All these existing mucoadhesive polymer-based systems exhibit lower adhesion strength compared to that of BC_PDz composites, which is around 15-22 kPa. Higher strength displayed by BC_PDz can be attributed to the covalent bond mechanism by carbene insertion depicted in
Apart from drug-loaded mucoadhesive system, other mussel inspired bioadhesive systems have been evaluated. It was reported wet adhesion through tannic acid-based bioadhesives exhibited adhesion strength ranging from 30-50 kPa. Though these bioadhesive exhibit high adhesion strength, the shear moduli exhibited by the crosslinked adhesives is as low as 10 Pa, which indicates the weak mechanical properties of the system. The gelation requires the addition of silver nitrate solution, and the minimum gelation time required is around 240 seconds, thus making the application of bioadhesive a complicated process. Another citrate-based mussel inspired bioadhesive was reported, wherein adhesive strength ranging from 30-160 kPa is reported upon crosslinking. However, crosslinking requires the addition of silver nitrate and sodium periodate, which are oxidative along with the requirement of high gelation time as 60-500 seconds. In another report, prepolymer liquid-based system which transformed into in situ bioadhesive amphiphilic systems was studied. The gels were formed when appropriate amount of poly(2-dimethylaminoethyl)methacrylate (PDMA) was added to prepolymer mixture of PEG and PCL. The gels exhibited storage modulus ranging from 50-200 kPa and adhesion strength of 40-90 kPa. However, the minimum gelation time required for the formation of gels was as high as 8 minutes. None of the reported studies provided any evidence of cohesive failure upon removal despite of the high adhesion strength, thus exposing the tissues to damage upon removal, if required. The present BC_PDz composite hydrogel exhibits on demand curing in 15-60 seconds, cohesive failure when removed, and requires no additional chemicals.
Advantages of aqueous BC_PDz mucoadhesive hydrogels are further discussed below.
BC_PDz hydrogel adhesive composites of the present disclosure have several advantages as compared to previously reported systems, thus proving its suitability for application as mucoadhesive system. Some of the advantages of the BC_PDz composites are (i) on-demand adhesion with crosslinking time as low as 15-60 seconds, (ii) covalent bonding mechanism for adhesion, (iii) storage modulus ranging from 1-10 kPa (measuring gap 0.45 mm) mimicking oral tissue, (iv) wet tissue adhesion strength of 15-22 kPa, (iv) cohesive failure in the composite matrix, (v) no harsh chemicals/oxidants required for crosslinking, and (vi) reduced concentration of PDz in the composite thus improving the biocompatibility. The present composites also exhibit adhesion strength similar to BC_PDz film (7-16 kPa) systems evaluated in examples 2A to 2S.
In conclusion, the aqueous adhesive BC_PDz hydrogel composites are formulated with the ability to adhere directly to tissue substrates in the oral cavity upon photocuring. BC hydrogel serves to improve the mechanical properties of the system employing nanofiber networks, which further form adhesive crosslinked matrix upon reaction with PDz on light exposure. Tunable adhesion strength ranging from 3-33 kPa against wet tissue mimic and 8-70 kPa against dry PLGA substrate is obtained. The adhesion strength is variable across the type of BC_PDz composite based on BC moles, PDz moles, and total solute percentage in the composite. The composites exhibited a change from viscous (liquid-like) state to elastic (solid-like) material properties as crosslinking progresses upon photocuring. The primary mechanism of adhesion is —OH insertion of carbenes generated upon photocuring followed by the formation of covalent bonds. Cohesive failure exhibited by the mucoadhesive patch prevents the damage of soft tissues in the oral mucosa upon removal. The aqueous BC_PDz composites forming covalent adhesion present a new tool towards mucoadhesive delivery systems targeting oral cavity diseases and lesions.
Example 4A: Present Mucoadhesive in the Form of Integrated Adhesive Patch Exhibit Adhesion Properties Superior to Marketed FormulationsExamples 4A to 4C are generally intended to demonstrate the present mucoadhesive configured in the form of an integrated adhesive patch instead of a film (examples 2A to 2S) or a hydrogel (examples 3A to 3H).
BC_PDz mucoadhesive patches are developed by coating BC films (freeze-dried (FD), press-dried (PD)) with varying moles (0.06-0.11 moles) of PDz bioadhesive (PDz integrated to about 40-50 μm thickness of BC). This provides an integrated system wherein the patch can directly be applied to the application site and photocured, unlike the 2-component system in the form of a film. The adhesive patch can also be stored in dry form, thus prolonging the shelf stability. The adhesive patches are evaluated for lap shear adhesion test and compared for their performance with marketed denture and medicated adhesives. It is observed that the patches exhibit significantly higher adhesion strength than control BC films in dry (D) and rehydrated states (Rh) (
Electrosurgery is used routinely in surgery to cut, coagulate, dissect, fulgurate, ablate and shrink tissue. High frequency (100 kilohertz to 5 megahertz), alternating electric current at various voltages (200-10,000 Volts) is passed through tissue to generate heat. In our recent investigation we evaluated BC_PDz adhesive patch and BC with other adhesives for electrosurgical activation. Other biomaterials like collagen were also evaluated. It is observed that collagen is malformed under electrosurgery and therefore cannot be used for electrosurgical activation (wherein collagen substrate was placed on the adhesive followed by electrosurgical activation), This behaviour can be attributed to protein denaturing that occurs in protein-based biomaterials and biomaterials with low heat tolerance. The activation of caproglue BC combination through electrosurgery was also investigated. It is observed that BC does not get deformed upon electrosurgery and activates the adhesive (liquid caproglue is converted to solid, observed upon scratching with tweezer) while preventing any damage to the tissue underneath. Similarly, the activation of BC_PDz adhesive patch using electrosurgery was investigated.
The adhesive patches have been loaded with various drugs with the drug loading efficiency as mentioned in
Structure Property Relationships of BC delivery vehicles have been studied with Valganciclovir.
BC films give tunable drug elution rates of 0.5 to 50 ug per hour per cm2.
BC films provide tunable drug release for 10-48 hours in sustained manner.
The burst release profile of BC films can be controlled to achieve 5-100% release in first 30 minutes.
Drug loaded films have also been evaluated for adhesion properties.
The preceding examples demonstrate that the present mucoadheisve can be configured to have at least 3 forms such as a film configuration, a hydrogel configuration and an integrative patch. The comparison of these forms are shown in
The present mucoadhesive includes bacterial cellulose (BC), which is a natural product derived from Gluconacetobacter species. The extracellular polymer absorbs 80-100 times water of its own dry weight with the inherent ability to self-regulate water absorption. The cellulose matrix reaches a hydration plateau, after which no more water is absorbed, and the cohesive properties are retained, even in excess moisture. Another attribute of the material properties is the amorphous structure that results in translucent to transparent properties in both the dry and wetted states, which can be exploited for optical tissue sensors, photochemistry, photocuring, optical characterization, or combination thereof. BC and other similar natural polymers are edible food product, for example translucent Nata de coco, and is generally regarded as safe (GRAS) by FDA. BC-based wound dressings have also been approved by FDA and marketed for burn wounds owing to its wound healing potential. In addition, the present mucoadhesive and method of manufacturing the composite mucoadhesive may include the following combination of characteristics/features that allows for extended fixation on wet substrates.
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- 1. Composition of matter that includes composites formulations with one self-regulating hydrophilic polymer, one stimuli-sensitive bioadhesives capable of bonding substrates, including but not limited to wet substrates, and up to 50% w/w incipients, active pharmaceutical ingredients, microorganisms, minerals, particles, biologic, or combination thereof.
- 2. Self-regulating water absorption that maintains cohesive material properties, prevent brittle fracture of the mucoadhesive by physiological stress within a 2-7 day programmed period.
- 3. Transparent or translucent optical properties of light wavelengths from 350 to 1100 nm.
- 4. Film forming patches that withstand physiological stresses on epithelial and mucosa surfaces for 2-7 days.
The present mucoadhesive can be configured for other medications or other purposes, e.g. nicotine oral patch, tissue engineering scaffold for chronic oral ulcers. The present self-regulating mucoadhesive platform has numerous industrial applications, including but not limited to, controlled chemical delivery, substrate sealants, fixation of medical implants, film-forming barriers (microorganisms, chemical, physiological fluid), air/liquid interface exchange, tissue reconstruction, or combination thereof. For example, as a buccal wound dressing, it has the following advantages:
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- 1) Provide wound dressing in wet oral environment through minimally invasive surgeries or robot-assisted surgeries.
- 2) Display non-inferiority to current buccal wound treatment options.
- 3) Display tissue fixation 10-100× times longer the oral gel or cream formulations.
- 4) Reduced dosing frequency and increase patient compliance.
The site of applications of the present mucoadhesive can include and are not limited to the gastrointestinal tract and mucosal tissues (e.g. eye, nasal cavity, reproductive systems). The present mucoadhesive can be used (i) to deliver active ingredients locally to the oral cavity for oral ulcers, lesions, and diseases, (ii) as a barrier patch for diseased tissues and wound protection, (iii) as an acid barrier in gastrointestinal acid reflux, (iv) prevent air/liquid exchange in pneumothorax, (v) for tissue reconstruction in transplanted or resected tissues, e.g. colon cancer, oesophagus transplant, and/or (vi) transmucosal drug delivery system, for example, anti-inflammatory medications for chemotherapy patients experiencing inflamed mouth sores.
While the present disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as defined by the appended claims. The scope of the present disclosure is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.
Claims
1. A mucoadhesive comprising:
- a bacterial cellulose; and
- a bioadhesive incorporated to the bacterial cellulose,
- wherein the bioadhesive comprises a polymer or a dendrimer, wherein the polymer and the dendrimer are grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus.
2. The mucoadhesive of claim 1, wherein the moiety comprises a diazirine, and the polymer or the dendrimer comprises polycaprolactone or a poly(amidoamine) dendrimer, respectively.
3. The mucoadhesive of claim 1, wherein the bacterial cellulose is a film comprising a network of cellulose nanofibers.
4. The mucoadhesive of claim 1, wherein the bacterial cellulose is dried or rehydrated.
5. The mucoadhesive of claim 1, wherein the bioadhesive is deposited on the bacterial cellulose.
6. The mucoadhesive of claim 1, wherein:
- the bacterial cellulose is a hydrogel; or,
- the bacterial cellulose is a hydrogel and the bioadhesive is present in an amount of 10 wt % to 35 wt % of the mucoadhesive.
7. (canceled)
8. The mucoadhesive of claim 1, wherein:
- the bioadhesive is included in the bacterial cellulose; or
- the bioadhesive is included in the bacterial cellulose and the bioadhesive is present in an amount of 15 wt % to 60 wt % of the mucoadhesive.
9. (canceled)
10. The mucoadhesive of claim 1, wherein the bacterial cellulose is of a species derived from Acetobacteraceae family.
11. The mucoadhesive of claim 1, wherein the bacterial cellulose is optically pervious to light having a wavelength ranging from 280 nm to 1100 nm.
12. The mucoadhesive of claim 1, wherein the stimulus comprises:
- light having a wavelength ranging from 280 nm to 500 nm; and/or
- a voltage ranging from −10 kV to 10 kV.
13. The mucoadhesive of claim 1, further comprising a pharmaceutical drug incorporated to the mucoadhesive.
14. The mucoadhesive of claim 1, further comprising a polymeric coating on the mucoadhesive.
15. A method of forming the mucoadhesive of claim 1, the method comprising:
- providing a bacterial cellulose;
- providing a bioadhesive comprising a polymer or a dendrimer, wherein the polymer and the dendrimer are grafted with a moiety which forms covalent interactions with a tissue surface in the presence of a stimulus; and
- incorporating the bioadhesive to the bacterial cellulose.
16. The method of claim 15, wherein providing the bacterial cellulose comprises:
- inoculating bacteria in one or more culture media;
- extracting raw bacterial cellulose from the one or more culture media; and/or
- treating the raw bacterial cellulose with an alkali to remove the bacteria from the bacterial cellulose.
17. The method of claim 15, wherein providing the bacterial cellulose comprises freeze-drying or press-drying the bacterial cellulose.
18. The method of claim 17, further comprising:
- rehydrating the bacterial cellulose which is freeze-dried or press-dried prior to incorporating the bioadhesive.
19. The method of claim 15, wherein providing the bioadhesive comprises:
- grafting a diazirine on a polycaprolactone or a poly(amidoamine) dendrimer; and
- mixing the polycaprolactone or the poly(amidoamine) dendrimer grafted with the diazirine in a buffer.
20. The method of claim 15, wherein incorporating the bioadhesive to the bacterial cellulose comprises depositing the bioadhesive on the bacterial cellulose.
21. The method of claim 15, wherein incorporating the bioadhesive to the bacterial cellulose comprises:
- (i) contacting the bacterial cellulose with an organic solvent comprising the bioadhesive; and
- removing the organic solvent to have the bioadhesive included in the bacterial cellulose; or
- (ii) mixing the bacterial cellulose and the bioadhesive to form a hydrogel.
22. (canceled)
23. The method of claim 15, further comprising coating a polymeric coating on the mucoadhesive.
Type: Application
Filed: Nov 30, 2021
Publication Date: Dec 7, 2023
Inventors: Sierin LIM (Singapore), Terry William Joseph STEELE (Singapore), Juhi SINGH (Singapore)
Application Number: 18/254,756